RNA molecules have the capacity to act as potent modulators of gene expression in vitro and in vivo. These molecules can function through a number of mechanisms utilizing either specific interactions with cellular proteins or base pairing interactions with other RNA molecules. This modulation can act in opposition to the cellular machinery, as with RNA aptamers that disrupt RNA-protein and protein-protein interactions, or in concert with cellular processes, as with siRNAs that act by redirecting the endogenous RNAi machinery to targets of choice. Modulation of gene expression via RNA effector molecules has great therapeutic potential as the modulatory complexes formed, be they RNA-protein complexes or RNA-RNA complexes, are often highly specific (Aagaard et al., 2007, Adv Drug Deliv Rev., 59:75-86; de Fougerolles et al., 2007, Nat Rev Drug Discov., 6:443-53; Grimm et al., 2007, J Clin Invest., 117:3633-411-4; Rayburn et al., 2008, Drug Discov Today., 13:513-21). When this specificity is determined by the well established rules of base pairing, targeting of this regulatory machinery to particular gene products becomes accessible to direct experimental design.
RNA molecules that modulate gene expression may take a number of different forms. Perhaps the seminal example for all is the antisense RNA molecule. This inhibitory RNA is typically a direct complement of the mRNA transcript it targets and functions by presenting an obstacle to the translational machinery and also by targeting the transcript for degradation by cellular nucleases. Another related and overlapping class is the small inhibitory RNA (siRNA) which acts through the post-transcriptional gene silencing or RNAi pathway. These RNAs are typically about 21-23 nucleotides in length and associate with specific cellular proteins to form RNA-induced silencing complexes (RISCs). These small RNAs are also complementary to sequences within their mRNA targets and binding of these complexes leads to translational silencing or degradation of the transcripts (Farazi et al., 2008, Development., 135:1201-145-7; Sontheimer et al., 2005, Nat Rev Mol Cell Biol., 6:127-38; Zamore et al., 2005, Science., 309:1519-24).
Two additional classes of RNA molecules that can modulate gene expression and activity are the catalytic RNA ribozymes and the competitive RNA aptamers. Ribozymes are RNA based enzymes that catalyze chemical reactions on RNA substrates, most often hydrolysis of the phosphodiester backbone. Formation of the catalytic active site requires base pairing between the ribozyme and the RNA substrate, so ribozyme activity can also be targeted to desired substrates by providing appropriate guide sequences (Wood et al., 2007, PLoS Genet., 3:e109; Scherer et al., 2007, Gene Ther., 14:1057-64; Trang et al., 2004, Cell Microbiol., 6:499-508). When targeted to mRNA transcripts, ribozymes have the potential to cleave those transcripts and lead to downregulation of the associated protein (Liu et al., 2007, Cancer Biol Ther., 6:697-704; Song et al., 2009, Cancer Gene Ther.,; Weng et al., 2005, Mol Cancer Ther., 4:948-55; Li et al., 2005, Mol Ther. 12:900-9). RNA aptamers are typically selected from pools of random RNA sequences by their ability to interact with a target molecule, often a protein molecule. Engineering RNA aptamers is less straightforward as the binding is not defined by base pairing interactions, but once an effective sequence is found the specificity and affinity of the binding often rivals that of antibody-antigen interactions (Mi et al., 2008, Mol Ther., 16:66-73; Lee et al., 2007, Cancer Res., 67:9315-21; Ireson et al., 2006, Mol Cancer Ther., 12:2957-62; Cerchia et al., 2005, PLoS Biol., 3:e1230). RNA aptamers also have a greater range of target molecules and the potential to alter gene activity via a number of different mechanisms.
Two methods for delivering inhibitory RNA molecules to cells have become standard practice. The first method involves production of the RNA molecules in the test tube by using purified polymerases and DNA templates or through direct chemical synthesis. These RNA molecules can then be purified and mixed with a synthetic carrier, typically a polymer, a liposome, or a peptide, and delivered to the target cells (Aigner et al., 2007, Appl Microbiol Biotechnol., 76:9-21; Juliano et al., 2008, Nucleic Acids Res., 36:4158-71; Akhtar et al., 2007, J Clin Invest., 117:3623-32). These molecules are delivered to the cytoplasm where they bind to their mRNA or protein targets directly or through the formation of modulatory complexes. The second method involves transfecting the target cells with a plasmid molecule encoding the biologically active RNA. Once again, the purified plasmid molecule is coupled with a synthetic carrier in the test tube and delivered to the target cell (Fewell et al., 2005, J Control Release., 109:288-98; Wolff et al., 2008, Mol. Ther., 16:8-15; Gary et al., 2007, J Control Release., 121:64-73).
In this case, the plasmid template must be delivered to the cell nucleus where the DNA is transcribed into the biologically active RNA molecule. This RNA is then exported to the cytoplasm, where it finds its way to modulatory complexes and specific mRNA transcript targets. With each of these approaches, the extent of gene regulation within a population of cells is limited by the transfection efficiency of the delivery system. Cells that are not transfected with the biologically active RNA molecules or plasmids encoding biologically active RNAs have no mechanism for receiving the modulatory signal. Although high transfection efficiencies are possible for cells growing in culture, achieving similar extents of transfection is difficult in vivo. This delivery issue is currently the major prohibitive factor for the application of RNA based therapeutics in vivo as it limits the extent to which a particular gene can be regulated in a population of cells. Thus, there remains a need to for an effective delivery system for efficiently delivering biologically active RNAs to cells and tissues.